Protein as a diagnostic of cancer

ABSTRACT

The presence of A-protein in an abnormally large amount in a sample, such as blood, from an individual is diagnostic of primary or metastatic cancer in the individual. The presence of A-protein is most readily detected by immunological reaction of it with specific antibodies. A preferred procedure for detecting the presence of A-protein in samples is by a sandwich assay using two antibodies with different epitopic specificities for A-protein.

RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationNo. PCT/US96/00098, filed on Jan. 10, 1996, which is a Continuation-InPart of U.S. Ser. No. 08/370,969, filed Jan. 10, 1995, now abandoned,which is a Continuation-in-Part of U.S. Ser. No. 07/858,841, filed Mar.27, 1992, now abandoned, which is a Continuation-in-Part of 07/802,370,filed Dec. 4, 1991 (now abandoned), which is a Continuation of07/170,737, filed Mar. 21, 1988 (now U.S. Pat. No. 5,100,661, issuedMar. 31, 1992), all of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

The diagnosis of cancer in individuals has remained a difficult task toaccomplish. Although some diagnostic markers are available that areassayable from blood or tissue samples, e.g. Carcinoembryonic Antigen(CEA), Alpha Fetoprotein (AFP) or Prostate Specific Antigen (PSA), theassays using these markers have not, to date, been markedly predictiveof the presence of cancer in these individuals, as verified by otherclinical diagnoses. The sensitivity and specificity of these assays hasbeen disappointingly low. Time-consuming and labor-intensive clinicalassessments (e.g. palpations, x-rays, mammograms, biopsies) haveremained the accepted methods for diagnosing cancer. Thus, a need existsfor a marker, preferably present in a biological sample, from anindividual, such as blood that is predictive of the presence of cancerin the individual. In particular, a need exists for the existence of amarker and an assay to measure the presence and amount of this markerfor individuals who have cancer in an early stage. If such a diagnostictest were available, early treatment with beneficial outcomes would bemore likely than at present.

It is an object of this invention to provide methods for detecting thepresence of primary or metastatic cancer in an individual that involvesthe detection of a cancer-diagnostic protein. It is also an object ofthis invention to provide compositions of matter that can be used todetect the presence of primary or metastatic cancer in an individual.

SUMMARY OF THE INVENTION

This invention pertains to methods of detecting cancer in an individualfrom the results of an assay of a biological sample, such as a bloodsample, from the individual. In this assay, the sample is incubated withat least one antibody that is immunoreactive with a cancer-diagnosticprotein that may be present in the biological sample. Theimmunoconjugates that are formed in the cancer-diagnosticprotein:antibody reaction are detected. The presence of an abnormallyhigh concentration of these immunoconjugates indicates that theindividual from whom the sample was taken has primary or metastaticcancer.

In a preferred embodiment of this invention, the cancer-diagnosticprotein is A-protein and the sample is incubated in a sandwich assay forA-protein. An antibody that is immunoreactive with A-protein is attachedto a solid support. The sample is allowed to immunoreact with theattached antibody and with a second antibody that is immunoreactive withanother region of A-protein (i.e., a region other than the regionimmunoreactive with the solid support-attached antibody). The resultanttwo antibodies-A-protein complex thereby forms a sandwich. The amount ofbound second antibody is detected. This amount of detected secondantibody is directly proportional to the amount of attached A-protein.An abnormally large amount of detected second antibody is indicative ofthe presence of primary or metastatic cancer that is being detected bythe assay of the biological sample.

Another embodiment of this invention is a test kit that contains one ormore antibodies to be used in the assay for the cancer-diagnosticprotein (e.g. A-protein). One of the antibodies is immunoreactive withone epitopic region of the cancer-diagnostic protein and, if a secondantibody is included, the second antibody is immunoreactive with anepitopic region of the cancer-diagnostic protein separate from theepitopic region that is immunoreactive with the first antibody. In apreferred embodiment of the test kit, there are two antibodies that areimmunoreactive with two epitopic regions of A-protein. One of theantibodies is attached to a solid support, such as the walls and bottomsof wells of a microtiter plate. The other antibody has a detection labelbound to it.

Yet another embodiment of this invention is one or more antibodies thatimmunoreact with a cancer-diagnostic protein, i.e. A-protein. Theseantibodies are used to detect or assess the presence of thecancer-diagnostic protein. The assessment can be performed usingbiological samples such as blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the chromatographic separationof soluble A-protein: FIG. 1A is an optical scan of the A_(s) form ofA-protein purified on a high pressure liquid chromatography (HPLC) gelfiltration column;

FIG. 1B is an optical scan of the HPLC-purified A_(s) rechromatographedon a DEAE-cellulose anion exchange column with a linear salt gradient(dashed line) to confirm the effectiveness of the purificationprocedure.

FIG. 2 is a photographic representation of the purification of the A_(m)and A_(s) forms of the A-protein by SDS-PAGE. The gel was stained withsilver. Lane 1 is purified A_(m); lane 2 is purified A_(s); lane 3 showsmolecular weight standards.

FIG. 3 is a graphic representation of the binding of the GTP-analogGMP-PNP by soluble and membrane-bound forms of A-protein in the presenceof adenosine nucleotides.

FIG. 4 is a graphic representation of the ATPase activity of A_(s) andA_(m) in the presence of GTPγS.

FIG. 5 is a scattergram which depicts the A-protein concentration inblood for control individuals and for individuals that have beendiagnosed as having metastatic breast cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to methods and compositions fordiagnosing the presence of primary or metastatic cancer in individuals.Primary cancer is cancerous growth that is confined to a particularanatomical region of the body or is composed of cells that formed theinitial cancerous lesion. Metastatic cancer is cancerous growth that hasspread from an original primary site in the body through either theblood or the lymphatic system, or both, and is growing at a site removedfrom the primary tumor; it is also recurrent disease spread secondary tothe treatment of a primary tumor.

The primary or metastatic cancer that can be diagnosed by employing themethods and compositions of this invention are any one of a largevariety of cancers. Included in this list of detectable cancers, by themethods and compositions of this invention, are breast cancer, prostatecancer, liver cancer, lung cancer, colorectal cancer, gastric tissuecancer, pancreatic cancer, bladder cancer, head and neck cancer,endometrial cancer, parotid cancer, cholangio cancer, kidney or renalcancer, cervical cancer, thyroid cancer, brain cancer, mouth cancer,uterine cancer, abdominal cancer, tongue cancer, lip cancer, analcancer, pelvic cancer, inguinal cancer, penile cancer, chest wallcancer, fallopian tube cancer, POEMS, lymphoma, leukemia, multiplemyeloma, melanoma and various sarcomas. These cancers, and most likelyother types of cancer, can be detected in various stages of theirprogression including stage 1 and stage 2. The detection of the presenceof these cancers can be distinguished from the presence of benign tumorsor the absence of cancer in individuals by using the methods andcompositions of this invention.

The methods of this invention preferably involve the use of antibodiesthat are immunoreactive with a cancer-diagnostic protein. The preferredcancer-diagnostic protein of this invention is A-protein. Followingincubation of one or more of the antibodies with a biological samplefrom the individual, the presence of an abnormally large amount ofantibody-A-protein immunoconjugate indicates the presence of primary ormetastatic cancer in the individual.

Blood is an often used and preferred biological sample. When blood isthe biological sample, the preferred blood constituent that is analyzedis either plasma or serum, more preferably serum. Since blood or otherbody fluids are preferred biological samples, the immunoreactivecancer-diagnostic protein, e.g. A-protein, is detected from a liquidmedium. When blood is the biological sample, the liquid medium iscirculating in the body. Thus, the methods of this invention do not relyon an analysis of localized, e.g. biopsy, material but, rather, can beemployed with a readily obtainable biological sample such as blood serumor plasma. Even when such blood sampling is employed, the variety ofcancers that can be detected is quite extensive.

In particular embodiments of the invention, the presence of aconcentration of A-protein in a biological sample from a particularindividual that is more than twice the concentration of A-protein foundin similar samples from individuals without cancer is indicative of thepresence of cancer in that particular individual. In other words, ifbiological samples of the same size are taken from individuals who arefree of cancer and the average (mean) amount of A-protein in thesesamples is determined, an individual is predicted to have cancer if abiological sample of the same size from that individual has more thantwice the amount of A-protein in it than was determined as the averageamount of A-protein for the cancer-free individuals. In these particularembodiments, the factor of 2.0 is considered to be a threshold value.Individuals are predicted to have cancer if concentrations of A-proteinin biological samples from them have more than 2.0 times that of theaverage concentration of A-protein for cancer-free individuals, i.e., ifthe ratio of the A-protein concentration from the tested individual'ssample to the A-protein concentration average from cancer-freeindividuals is greater than 2.0. Ratios of tested individual's A-proteinconcentration to the cancer-free individuals' A-protein concentrationaverage of less than 2.0 indicates that the tested individual does nothave cancer. The threshold value of 2.0 is a particularly preferredvalue. Other threshold values can be established for predicting thepresence of cancer in an individual. These threshold values will dependon the accuracy and reproducibility of the particular cancer diagnosticassay as well as the predictive reliability that is sought for theassay.

The actual amount or concentration of A-protein in a biological samplefrom an individual can have diagnostic or prognostic value. Bydetermining the quantity of A-protein over a period of time for anindividual, it is possible to monitor the progressiveness of the cancer(as the quantity of A-protein increases) or the regression of thecancer, e.g. as a result of therapeutic treatment (as the quantity ofA-protein decreases). The quantity of A-protein that is detected can bemeaningful as an indicator of the presence and vigor of cancer growth.The A-protein quantity is monitored in relation to a baseline value forthat individual or in comparison to the average amount of A-protein insimilar biological samples from cancer-free individuals. When theA-protein quantity is greater than a particular value, established bythe monitor of the assay, the individual is considered to have cancer.The actual A-protein quantities in excess of this established valueindicate the severity of the cancer and lesser A-protein quantities,with or without therapeutic treatments, indicate a diminution of cancerseverity.

In other particular embodiments of the invention, the presence of morethan about 10 ng of A-protein per ml of blood is predictive ofmetastatic cancer. In these methods, assays for metastatic cancer arealso based on measurements of the amount of A-protein in biologicalsamples (e.g., fluid samples such as blood plasma or serum, urine, etc.)from an individual. Again, the amount of A-protein in the biologicalsample is indicative of the presence or absence of metastatic cancer inthe individual from whom the sample was obtained. An abnormally largeamount of A-protein indicates the presence of metastatic cancer; anormal amount of A-protein indicates that the individual does not havemetastatic cancer.

The usefulness of these assays is readily apparent: a relatively simpleassay is predictive of the presence or absence of primary or metastaticcancer.

A variety of techniques are known and available to the artisan forassaying for the amount of A-protein in a biological sample. Thesetechniques include isolating and quantifying the A-protein in the sampleby solvation and centrifugation procedures, by column chromatography orgel electrophoretic separation procedures, by filtration procedures, byenzymatic recognition procedures, or by binding procedures withmolecules that recognize and bind A-protein with specificity such asaffinity chromatography or immunoassay. Such specific binding processescan also be used to detect and quantify the A-protein in situ inbiological samples by assessing the volume or area distribution of thespecific binding molecule in the sample.

A preferable technique for assaying for the amount of A-protein in abiological sample is immunological recognition with antibodies thatspecifically bind A-protein or particular epitopes of A-protein. Suchantibodies are compositions of the present invention. These antibodiesselectively recognize A-protein determinants and bind to thesedeterminants with high affinity. The antibodies can be used singly asaffinity immobilization or as tagging binding moieties in proceduressuch as Western blot or electrophoretic pattern analyses, or multiply tobind to different A-protein determinants or epitopes such as in sandwichassays. These antibodies can have substances that act as labels attachedto them for ease of identification following binding of the antibody toA-protein. The antibodies of this invention bind to A-protein withspecificity so A-protein or specific epitopes of A-protein can bedetected with particularity.

Antibodies which can be used within this invention are reactive withA-protein. The term antibody is also intended to encompass bothpolyclonal and monoclonal antibodies. The term antibody is intended toencompass mixtures of more than one antibody reactive with A-protein(e.g., a cocktail of different types of monoclonal antibodies reactivewith A-protein). The term antibody is further intended to encompasswhole antibodies, biologically functional fragments thereof, singlechains or single chain fragments with A-protein binding properties, andchimeric antibodies comprising portions from more than one species,bifunctional antibodies, etc. Biologically functional antibody fragmentswhich can be used are those fragments sufficient for binding of theantibody fragment to A-protein.

The chimeric antibodies can comprise portions derived from two differentspecies (e.g., human constant region and murine variable or bindingregion). The portions derived from two different species can be joinedtogether chemically by conventional techniques or can be prepared asfusion proteins using genetic engineering techniques. In addition, DNAencoding the proteins of both the light chain and heavy chain portionsof the chimeric antibody can be expressed together as fusion proteins.

It has been determined that particular intracellular molecules, calledA-proteins, are intimately involved in the regulation of theinositol-related signal transducing system. In this system, A-proteinsfunction by activating phospholipase C (PL-C) to generate the secondmessengers inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DG).Upon stimulation of a membrane bound receptor, an A-protein binds withGTP to form an intermediate which functions to activate PL-C. When theGTP of the intermediate is hydrolyzed to GDP, PL-C activationterminates. A-proteins are accordingly important G-type signaltransducing molecules critical to proper functioning of the cellularinositol metabolic pathway.

These signal transducing molecules are useful in producing theantibodies that are used in the foregoing methods of the presentinvention. The signal transducing molecules that are used to produce theantibodies are isolated and purified A-proteins and epitopic fragmentsof A-protein.

The cancer-diagnostic proteins of the invention, e.g. A-protein, canalso be useful in immunotherapy. When these proteins are introduced intothe blood or lymphatic system, they can stimulate the immune system torespond, i.e. to recognize the presence of cancer in the host and torespond to it. Thus, by introducing these cancer-diagnostic proteinsinto the individual's bloodstream or lymphatic system from externalsources, the immune system can be stimulated to generateimmunosuppressive activity to eradicate the cancer.

In addition, the antibodies of this invention can have therapeuticvalue. When they are administered to an individual, they can immunoreactwith the cancer-diagnostic protein, e.g. A-protein, and thereby initiatefurther immune system response, e.g. by stimulating the production ofT-cells, to eliminate the cancer cells present in the individual.

The term “A-protein” was originally used to describe a rod photoreceptorprotein of approximately 20 kilodaltons molecular weight based on theelectrophoretic mobility of this protein through a gel under reducingconditions. From, an analysis of the protein as an expressed product ofthe known A-protein gene, the molecular weight of the A-protein is 26kilodaltons. Improved extraction and separation methods combined withpreliminary sequence data on the separated forms indicates that theentity referred to previously as A-protein may consist of at least twostructurally and functionally related proteins; one membrane-bound andone soluble. On this basis, the terminology used reflects the presumedin vivo state: A_(m), membrane bound (20 kD); and A_(s), soluble (19kD), again based on electrophoretic mobility under reducing conditions.These A-proteins include the amino acid sequences for the N-terminalregions set forth in the Sequence Listing as:

GNSKSGALSKEILEELQ (SEQ ID NO:1) (A_(m)); and

MGNSKSGALSKEILEELQ (SEQ ID NO:2) (A_(s)).

Further characterizations of the A-protein related molecules are thatthey comprise a single polypeptide chain with a significantlyhydrophobic region. These molecules also have the ability to bind andhydrolyze the nucleotides adenosine and guanosine triphosphate, and havethe ability to activate phospholipase C, phospholipase D, and possiblyalso phospholipase A₂, in the presence of GTP.

A-proteins can be isolated from mammalian (bovine) and amphibian (frog)rod outer segments (ROS) of photoreceptor cells of the eye byextraction, centrifugation, chromatography and other proteinpurification techniques known to those skilled in the art. Otherproteins with similar or identical physical and functionalcharacteristics as the A-proteins have been isolated from various othertissues from vertebrates and invertebrates. These findings indicate thatthe structure of the A-proteins has been conserved through evolution.A-proteins are quite labile in aqueous solution, but can besignificantly stabilized if disposed in aqueous solutions containing anonionic surfactant. They have a molecular weight in the range of 19 to20 kD, as inferred by comparison to molecular weight standards duringelectrophoretic separations under reducing conditions. Preferred methodsof isolating the native protein are disclosed in detail below. Goodpurification results have been achieved using filters with molecularweight cutoffs in the range of 10 kD and 30 kD.

A-proteins, various truncated or mutein analogs thereof, and fusedproteins comprising an A-protein and other protein domains can beproduced by various synthetic and biosynthetic means. For example, anappropriate host cell such as a microorganism, yeast, or eucaryotic cellculture can be genetically engineered to express an A-protein, or aportion or analog thereof. This may be accomplished by nowwell-established recombinant DNA technologies known to those skilled inthe art. The recombinant procedure may include the isolation orsynthesis of the gene encoding an A-protein, a portion, or analogthereof, and the integration of that gene into a plasmid. The amino acidsequences of the A-proteins may be established readily. The SequenceListing sets forth the N-terminus amino acid sequence of two forms ofA-protein (A_(m) and A_(s)) as SEQ ID NO:1 and SEQ ID NO:2,respectively. Gene synthesis from synthetic oligonucleotides and knownmutagenesis techniques provide the technologies to prepare an array ofanalogs, truncated A-protein forms, and fused proteins comprisingA-protein or an antibody binding domain thereof. Production of suchmaterials further may include the transformation of an appropriate hostcell with a vector harboring the recombinant DNA that includes the geneencoding A-protein or a portion or analog thereof, culturing thattransformed host cell, and isolation of the expressed protein. Given theavailability of A-protein-rich samples producible as disclosed herein,the recombinant production of the native form and various portions andanalogs thereof is well within the current skill in the art.

Alternatively, at least portions of the protein can be producedsynthetically by chemically joining amino acids in the correct sequence.

The isolated A-proteins, or portion or analog thereof, can be used asantigens to produce antibodies that are useful to detect A-protein influid samples from individuals, thereby assaying for the presence ofprimary or metastatic cancer. The antibodies can be part of a polyclonalantisera, or the binding portions thereof of these antibodies, raisedagainst A-protein, and shown to react with A-protein or with itsanalogs, fragments or to a particular epitope on A_(m) or A_(s). Theantibodies can be polyclonal or monoclonal antibodies produced bymethods known per se. The antibodies preferably are selected so as notto cross-react with other cellular components. The antibodies can be ofany class and subclass as determined by the Ouchterlony double diffusiontest. Antibodies of the IgG class are preferred. Alternatively,antibodies which recognize A-protein can be synthesized by biosyntheticor recombinant means, either in whole or in part.

In addition, the antibodies can be linked to other functional moleculessuch as toxins, fluorescent or absorption dyes, enzymes, or radioactivemarkers. In preferred embodiments, the antibodies are linked to biotinmolecules which have a particularly strong avidity for avidin orstreptavidin which, in turn, can be linked to fluorescent, absorption orradioactive markers. The linked antibodies can be used to detectA-protein from biological samples and thereby assay for primary ormetastatic cancer. The antibody-marker complex can be prepared bychemical linkage or by recombinant DNA techniques if the marker isproteinaceous.

The antibodies can also be labelled with a reagent which enables themonitoring or imaging of the antibody immediately after itsadministration to a patient. The label can be, for example, aradioisotope such as ¹²⁵I or ^(99m)Tc, both of which can be imagedextracorporeally by radiation detection means such as a gammascintillation camera. Alternatively, the antibody can be labelled with anon-radioactive, paramagnetic contrast agent capable of detection in MRIsystems. In such systems, a strong magnetic field is used to align thenuclear spin vectors of the atoms in a patient's body. The field is thendisturbed and an image of the patient is read as the nuclei return totheir equilibrium alignments. In the present invention, antibodies canbe linked to paramagnetic materials such as gadolinium, cobalt, nickel,manganese or iron complexes, to form conjugate diagnostic contrastreagents that are imaged extracorporeally with an MRI system.

If the antibodies are not linked to functional markers, conjugates ofantibody-A-protein can still be detected by using standard biochemicaltechniques to recover immunoprecipitates, such as centrifugation.

Anti-A-protein monoclonal antibodies can be obtained from hybridoma celllines formed upon the fusion of mouse yeloma cells with spleen cells ofmice previously immunized with A-protein that has been purified, forexample, from bovine ROS. The immunogen alternatively can be aderivative of A-protein, or an analog or portion thereof, produced invitro according to known mechanical or manual procedures of peptidesynthesis. Alternatively, the immunogen (A-protein) can be synthesizedby biosynthetic means using recombinant DNA technologies known to thoseskilled in the art. The mice whose spleen cells are chosen for fusionare preferably from a genetically defined lineage such as Balb/C. Themyeloma cells used in the fusion are from a mammalian,antibody-producing cell line, but most preferably are from a mouse cellline such as, e.g., NS-1. The monoclonal antibodies can be obtained fromascites fluid of mice injected with the fusion product.

In preferred embodiments of the invention, an immunogen for theproduction of polyclonal or monoclonal antibodies is a peptide with anamino acid sequence of approximately 16 amino acids taken from the knownamino acid sequence of A-protein. This immunogen is a peptide of 10-18amino acids in length. In particularly preferred embodiments, theimmunogen is either the 14 amino acid or 16 amino acid carboxyl terminusof A-protein, the peptide containing amino acids 60-71 of A-protein, thepeptide containing amino acids 142 to 158 of A-protein or the peptidecontaining amino acids 158-170 of A-protein. When this immunogen is usedto produce antibodies for incorporation in a sandwich assay, a secondimmunogen from A-protein is chosen other than the approximately 16 aminoacid peptide of the first immunogen. Another technique for obtainingantibodies that immunoreact with the second immunogen of A-protein is toimmunize an animal with intact A-protein and select antibodies thatimmunoreact with an epitope of A-protein other than that of the firstimmunogen. The production of two sets of antibodies with the propertiesof immunoreacting with different epitopes ensures that sandwich assaysusing these antibodies will not be impeded by competition of theantibodies for the same antigenic site. This feature increases thesensitivity of the assay for detecting the actual amount of A-proteinpresent in the sample.

The polyclonal or monoclonal antibodies so produced by known proceduresare specific for A-protein, and therefore are particularly useful forassaying for A-proteins.

In the present invention, either intact A-proteins or detectablefragments of A-proteins can be assayed by the disclosed methods. Theessential feature of the intact protein or detectable fragment thereofis the ability of these proteins or peptides to be detectable, i.e. toimmunoreact with immobilization or detection antibodies. Formation ofthe protein or fragment complexes with the antibodies and detection ofthese complexes are all that is required. Such detectable complexes canbe formed with A-protein fragments.

This invention includes a particular method for detecting the amount ofA-protein in a biological sample such as blood serum or plasma. In thismethod a first antibody which binds to a first epitope on A-protein isadhered to a solid support. The adhered first antibody is contacted withthe biological sample to be tested. Either simultaneously orsequentially thereafter, a labeled second antibody which binds a secondepitope on A-protein is added to the first antibody-biological samplemixture, thereby forming a first antibody:A-protein:second antibodyimmunocomplex attached to the solid support. This second antibody isattached to a detectable marker. Any unbound second antibody is removed,and the presence and, if desired, the amount of the marker is thendetected, its presence and amount being indicative of the presence andthe amount of A-protein in the sample. An abnormally large amount ofdetected A-protein, by such an assay, indicates that the individual fromwhom the biological sample was obtained has a high probability of havingprimary or metastatic cancer. In other words, this assay is diagnosticfor primary or metastatic cancer.

Test kits are also embodiments of this invention. These test kitscontain one or more of the polyclonal or monoclonal antibodies that areneeded to perform assays for the cancer-diagnostic protein, e.g.A-protein, that may be present in the biological samples, such as blood,obtained from individuals. These test kits can also contain the solidsupports, such as microtiter trays, for performing the assays.Instructions for performing the assays for A-protein can also beincluded in the kits. If desired, an identification label can beattached to an antibody of the test kits. In preferred embodiments ofthe test kits, antibodies are provided that allow sandwich assays to beperformed where the antigen is A-protein. In particularly preferredembodiments of the invention, one of the sandwich antibodies isunlabeled and adhered to a solid support. The other antibody has a labelbound to it for detection purposes.

The following examples further disclose the nature of the invention,without limiting the scope thereof.

EXAMPLE 1

Purification of Soluble (A_(s)) and Membrane-Bound (A_(m)) A-Protein

A-proteins were isolated from the retinas of cow eyes essentially asdescribed by Schmidt et al. (J. Biol. Chem, 262:14333-14336 (1987)).Bovine (cow or calf) eyes were obtained from a local abattoir within 2hours of killing. Bovine eyes were kept on ice in the dark for 30 to 60minutes. Retinas were dissected out and placed in buffer A (130 mM NaCl,20 mM Tris-HCl, pH 7.0; 1 ml per calf retina or 2 ml per cow retina).Gentle, repeated inversions of the container liberated large numbers ofROS into the buffer. The mixture was poured through a Buchler funnel toremove the retinas. The filtrate was allowed to settle in aconical-bottomed tube on ice for 5 minutes, allowing gross particulatematter to settle out of the ROS suspension. The supernatant was found,by means of microscopic examination on a hemocytometer, to consist ofgreater than 95% ROS. The ROS were disrupted with shear which wascreated by repeatedly drawing the suspension into the syringe andforcing it out against the wall of the container.

To separate particulate and aqueous fractions, the suspension wascentrifuged at 10,000 to 12,000×g for 20 minutes at 4° C. The pelletcontaining ROS membranes was washed once with a volume of buffer A equalto that of the removed supernatant. The resulting pellet was resuspendedin 3 to 6 ml of buffer T ((0.05%) Tween 20/80 (1:1) in double-distilledwater) and spun at 15,000×g for 45 minutes. The above manipulations werecarried out under dim red light. Both A-protein solutions (thesupernatants containing soluble and membrane-bound A-protein,respectively) were filtered through Centricon 30 microconcentrators(molecular weight cut-off of 30 kD, Amicon Corporation), withcentrifugation at 5,000×g in a refrigerated centrifuge. Theultrafiltrates were then concentrated and dialyzed in Centricon 10's(molecular weight cut-off of 10 kD) at 5,000×g.

The retentates contained proteins of 10 kD to 30 kD, with averageconcentrations of 100 to 200 μg/ml for soluble A-protein (A_(s)) and 30to 100 μg/ml for membrane-bound (A_(m)), reduced to a volume of 0.5 to 1ml. The purification of A_(s) resulted in a 320-fold enrichment andA_(m) was purified 20-fold. If the soluble A-protein solution was to bekept overnight before concentration and use, buffer A with 0.05% Tween20/80 (1:1) was added 1:5 to minimize aggregation of proteins in theconcentrated solution.

The A-proteins, purified by ultrafiltration as described above werefurther purified for sequence analysis by HPLC on a Bio-Sil SEC-125column in buffer A (A_(s)) or buffer T (A_(m)). Elution was isocratic.Pooled peaks were concentrated, dialyzed against water and lyophylizedprior to analysis.

For the purpose of confirming the purity of soluble A-protein used inexperiments, A_(s), purified by ultrafiltration as described, was run ona HPLC size exclusion column (TSK-2000, Bio-Rad) in buffer A (FIG. 1A),and then rechromatographed in 0.1 M potassium phosphate buffer on a DEAEanion-exchange column. Protein was eluted with a 0 to 200 mM NaClgradient (FIG. 1B).

The estimation of molecular weights of A_(m) and A_(s) were made fromthe relative mobility of each on SDS gels (see Example 2 and FIG. 2) anda calibrated gel filtration column. The agreement of weightdetermination between the native forms from the column and the denaturedforms on gels indicates that A-protein exists in vivo as a monomer.

The purification of A_(s) as described above results in preparations ofgreater than 95% purity. Any protein contaminant of the purified ASpreparations has been shown not to interact with guanosine or adenosinenucleotides under any of the conditions tested. Since the extractionsare sequential, A_(m) is purified to essential homogeneity by theprocedure described with no detectable contaminants, as shown in the SDSgel described below in Example 2 and pictured in FIG. 2, lane 1.

Once purified, the stability of the A-proteins differs markedly inaqueous solution. A_(m) is metastable in the purified state and retainsmost of its functional properties for several days at 4° C. A_(m) canalso withstand freezing and thawing in detergent without losing morethan 15 to 20% of its original activity. In contrast, A_(s) is labileunder a variety of conditions and no satisfactory methods of treatmenthave been found to prevent greater than 80% activity loss over a 48 hourperiod of 4° C. The soluble A-protein is markedly thermo- andcryolabile. Purified A_(s) loses activity rapidly at room temperature(half life=2 hrs) and freezing results in loss of virtually allactivity, presumably due to denaturation and/or aggregation. Thepurified soluble form aggregates readily in the absence of detergenttreatment and will precipitate overnight in the refrigerator under thoseconditions.

EXAMPLE 2

Gel Electrophoresis of Soluble (A_(s)) and Membrane-Bound (A_(m))A-Protein

Polyacrylamide gel electrophoresis was performed according to amodification of the methods of O'Farrell (J. Biol. Chem., 250:4007(1975)) in the presence of 0.1% SDS in a pore gradient gel (10 to 20%).Samples were applied in a sample buffer of 0.33 mM DTT, 7% SDS, 17%glycerol and 0.5 M Tris-HCl, pH 6.8, and run to equilibrium. Sampleswere not heat-denatured, in order to avoid the appearance of additionalbands caused by the formation of stable polymers. Proteins werevisualized with the Bio-Rad silver stain kit. Gels were calibrated usingpre-stained molecular weight standards from Bio-Rad (range 17 to 94 kD).

As shown in FIG. 2, “A-protein” includes A_(m), a membrane bound formhaving a molecular weight of about 20 kD and A_(s), a soluble formhaving a molecular weight of about 19 kD when the gels are run underreducing conditions. The soluble protein resolves into two closelyspaced bands on gels. The membrane-bound form migrates as a single band.

EXAMPLE 3

Production of Murine Polyclonal and Monoclonal Antibodies to A-Protein

Balb/c mice (6-8 weeks old; The Jackson Laboratory, Bar Harbor, Me.)were immunized with four injections of A-protein. The injections wereperformed one week apart and 50 μg A-protein (either A_(s), A_(m), orboth) was injected on each occasion. The first three injections weregiven intraperitoneally, and the fourth intravenously. A-protein wasinjected with complete Freunds adjuvant on the first occasion,incomplete adjuvant on the second and third occasions, and withoutadjuvant on the last occasion. Serum withdrawn prior to the lastinjection showed prominent binding to purified A-protein using a solidphase microtiter plate enzyme-linked immunoassay. The mouse with thebest immune response was sacrificed three days after the last injection.Lymphocytes from this animal were maintained as a polyclonal hybridomaby subcloning them as antibody producer cells in a Cellco bioreactor.These producer cells were maintained in liquid nitrogen cryostorage. Themurine polyclonal antibodies from these producer cells displayedspecificity for A-protein.

Monoclonal hybridomas were produced by fusion of spleen cells from thesacrificed mouse with NS-1 (P3NS-1/1-Ag4-1) myeloma cells (American TypeCulture Collection, Rockville, Md.; Accession No. TIB18). The method ofNadakavukaren (Differentiation, 27:209-212, (1984)) was employed toperform the fusions. Resultant clones were tested for binding toA-protein. Subcloning by serial dilution was carried out on one clone.The most productive subclones were injected into the peritoneal cavityof Balb/c mice to produce ascites fluid containing monoclonal antibody.The ascites fluid which was obtained was centrifuged, tested foractivity, and then stored at −70° C. until required.

The resulting 18 anti-A-protein antibodies were screened for antibodyisotype by the Ouchterlony double diffusion test in agar plates againstanti-IgM, anti-IgG, anti-IgG1, anti-IgG2a, anti-IgG2b, and anti-IgG3antibodies (Cappell). The results are shown in TABLE 1.

TABLE 1 mAb name isotype mAb name isotype 3A9A5 IgG 2a 3A9F2 IgG 2a3A12D7 IgM 3A12E9 IgM 4C5B4 IgM 4C5F3 IgM 5E6B1 n.d. 5E6H5 n.d. 7E4F9n.d. 7E4H12 n.d. 9D8F8 IgG 2a 9D8G11 IgG 2a 1B9B2 IgG 1 3A7G6 n.d.3B6C12 IgG 2a 3B9E1 IgM, IgG (mixed expression) 3F5H11 n.d. 4C8B2 n.d.“n.d.”—not determined

EXAMPLE 4

Assays for GTP and ATP Binding and Hydrolysis by A-Protein; ProteinAmount; and A-Protein Presence

A. GTPase and ATPase Assays

The rate of hydrolysis of GTP or ATP by both soluble and membrane-boundA-protein was assayed in a total volume of 200 μl. 1.0 to 10.0 μg A_(s)or A_(m), 5×10⁻⁵ M GTP or ATP, 67 to 335 nM [γ-³²P] GTP (2.8 Ci/mmol) or88.8 to 177.5 nM [γ-³²P] ATP (2.8 Ci/mmol) and 20 μl of stripped ROSmembranes (in the case of A_(m)) were mixed in buffer J (20 mM Tris-HCl,pH 7.0, 0.1 mM EDTA). The stripped ROS membranes (containing rhodopsinas the receptor) were prepared by washing ROS membranes 3 times inbuffer C(100 mM NaCl, 20 mM Tris-HCl, pH 7.0, 1 mM MgCl₂) and 3 times inwater containing 0.01% polyoxyethylene 23 lauryl ether (Brij 35 nonionicdetergent, Sigma Chemical Co.). The stripped membranes were resuspendedin buffer D (10 mM Tris-HCl, pH 7.0, 0.1 mM EGTA) prior to use.

The effect of light on the hydrolysis of GTP or ATP was investigated bymeans of duplicate incubations in light exposure trials (a bright 10msec xenon flash (Nikon) delivering 1.8×10³ μWcm⁻²sec⁻¹ which wassufficient to bleach greater than 70% of the rhodopsin present in eachsample) or kept in the dark as controls. Samples were incubated at roomtemperature for 5 minutes and quenched with 200 μl ice-cold quenchbuffer (50 nM KH₂PO₄, 6% Norite A, 10% TCA). The samples were kept onice for 30 minutes and spun down for 5 minutes in a microcentrifuge. 50μl aliquots of each supernatant were placed in a vial with 5 mlscintillation fluid and assayed for radioactivity. The hydrolysis of[γ-³²P]-GTP was measured in the presence and absence of photolyzedrhodopsin.

The GTPase activity of A_(m) was enhanced in the presence of theactivated receptor. In contrast, no effect on the hydrolysis rate wasobserved when unphotolyzed rhodopsin was added to the incubation in thedark. In the absence of rhodopsin or the presence of unbleachedrhodopsin, A_(m) had negligible GTPase activity.

On a mol/mol basis, the rate of GTP hydrolysis by A_(m) (0.458 GTPsec⁻¹/A_(m)) is comparable to that of transducin (0.512 GTP sec⁻¹/Tα)when both are measured at submaximal velocity in the presence ofphotoactivated rhodopsin. The GTPase rates for A_(m) and transducin areadditive when the two purified proteins are present in approximatelyequimolar concentrations.

Under all experimental conditions tested, the rate of GTP hydrolysis(GTPase activity) by purified A_(s) was insensitive to the presence ofbleached or unbleached rhodopsin.

The apparent Michaelis constant was determined for A_(s) and A_(m) bymeasuring the rate of GTP hydrolysis over a thousand-fold range ofsubstrate concentrations. K_(m) and V_(max) were determined byconstruction of double reciprocal plots and regression analysis. Dataare presented as mean±S.D. The results are given in TABLE 2.

TABLE 2 Values For GTPase (n) ATPase (n) K_(m):A_(S) 2.06 ± 2.46 × 10⁻⁶M(4) 1.50 ± 1.47 × 10⁻⁴M (3) K_(m):A_(m) 2.39 ± 1.87 × 10⁻⁶M (3) 1.44 ±0.56 × 10⁻⁵M (3) V_(max):A_(S) N.D. 0.56 (±0.09) pmol/mg A-protein min⁻¹V_(max):A_(m) 16.4 nmol/mgA_(m)min⁻¹ 2.90 (±0.16) pmol/mg A-proteinmin⁻¹

In the presence of rhodopsin the K_(m) values for A_(m) and A_(s) aresimilar indicating similar affinities for GTP.

A_(m) and A_(s) were found to have ATPase activity that was not receptorcoupled. The K_(m) values for both A_(m) and A_(s) ATPases are given inTABLE 2, above. Comparison of the rate constants of A_(m) indicates thatits affinity for ATP is approximately an order of magnitude greater thanthat of A_(s). The relative K_(m) values for GTPase and ATPase activityof both A_(m) and A_(s) indicate that GTP is the preferred substrate forbinding and hydrolysis.

The addition of rhodopsin to incubations did not enhance the rate of ATPhydrolysis of either protein. The ATPase rate declines slightly in thepresence of the activated receptor.

B. GTP Binding Assays

Assays of the binding of the GTP-analogs Gpp(NH)p and GTPγS (New EnglandNuclear) by both A_(m) and A_(s) were performed according to the methodsof Northup et al. (J. Biol. Chem., 257:11416-11423 (1982)). Binding wascarried out in a total volume of 100 μl of solution containing 5 to 10μg purified A-protein, 15.3 μM ³H-Gpp(NH)p (10 μCi) or 1.32 μM GTPγS³⁵(1 μCi) and buffer (100 mM NaCl, 0.1 mM EDTA, 20 mM Tris-HCl, pH 7.0).The samples were vortexed and incubated at 25° C. for 30 minutes,quenched with 200 μl ice-cold buffer (0.5 M NaCl, 0.1 M Tris-HCl, 0.1%Tween 80), and kept on ice for 30 minutes. The samples were placed ontonitrocellulose filters that had been previously washed with. 2 ml of thesame buffer. The filters were rinsed 5 times with 2 ml ice-cold bufferand assayed for radioactivity.

A_(m) bound both GTPγS and Gpp(NH)p spontaneously during a briefincubation at room temperature. The results are shown in TABLE 3 below.

TABLE 3 Form of Gpp(NH)p GTPγS Analog/mol A-protein mol GTP ProteinA_(m) 0.57 ± 0.12 0.93 A_(s) 0.29 ± 0.08 0.47

This process apparently required no cofactors. A_(m) bound less thanstoichiometric amounts from each GTP analog under the experimentalconditions used (TABLE 3). A_(s) bound significantly less of theseanalogues on a mol/mol basis after a similar incubation in the absenceof cofactors. GTPγS was more readily bound than Gpp(NH)p by both A_(m)and A_(s).

C. ATP/GTP Competition

The effect of adenosine nucleotides on the binding of Gpp(NH)p to amixture of A_(m) and A_(s) was examined because of the ability of theA-proteins to bind and hydrolyze ATP. Purified A_(m) and A_(s) weremixed (1:2), preincubated with ATP or ADP, and assayed for Gpp(NH)pbinding after a brief incubation by the rapid filtration method. Asshown in FIG. 3, ATP was an effective inhibitor of binding at allconcentrations tested. ADP was inhibitory in a concentration-dependentmanner at higher concentrations.

In contrast to the effect of adenosine nucleotides on the binding ofGpp(NH)p, they had negligible effects on the hydrolysis of GTP byA-protein. However, at micromolar concentrations, GTPγS was found tosignificantly inhibit the hydrolysis of ATP during the course of a onehour incubation (FIG. 4). GTP has a similar but less pronounced effecton ATP hydrolysis, indicating that both nucleotides compete for the sameor closely related binding sites on A-protein. These results alsosupport the finding that GTP is a more effective competitor than ATP forA-protein binding.

D. Protein Assays

Protein concentrations were determined according to Bradford (Anal.Biochem., 72:248 (1976)), using the Bio-Rad Microassay (CoomassieBrilliant Blue G-250). Bovine serum albumin was used as a standard.

On the basis of protein assays performed on the purified species, thecomplement of A_(m) relative to A_(s) (A_(m)/A_(s)) is 0.47. The ratioof the amounts of the separated soluble species recovered from highpressure columns (as estimated by optical density at 280 nm) and slabgels (as estimated by densitometry) is approximately unity. Thisindicates that all forms of A-protein are extracted in equivalentamounts from rods if the two soluble forms are the products of separategenes. If not, the membrane-bound form would thus be present at half theconcentration of the soluble.

E. Assay for A-Protein

The antibodies of this invention produced against A-protein were used tocreate an assay for the detection of that antigen in the serum ofhumans. The procedure employs an enzyme-linked immunosorbent assay(ELISA). A specific version of this assay is presented below in Example8.

The type of ELISA being used in this context was of the “sandwich”variety. This assay requires two different antibodies that are specificfor A-protein, where each antibody recognizes and binds to differentepitopes on the protein. One of the antibodies serves as the “capture”agent and was used unmodified to coat the bottom of a chamber in astandard 96-well microtiter plate. The unused portion of this antibodywas then removed from the well and a blocking agent (1% bovine serumalbumin (BSA)) was then placed in the chamber to block non-specificbinding sites. Serum from the propositus was then incubated in theprepared well for from 1 to 12 hours, and then removed. The well waswashed with a detergent solution, and the second antibody in solutionwas added to the well. The second antibody used in this procedure isphysically linked to a labeling substance such as an enzyme; forexample, the enzyme could be horseradish peroxidase (HRP). Followingthis incubation, the second antibody was removed and the well was washedonce more. The final step consisted of adding an appropriatecalorimetric substrate.

The specificities of these antibodies for different epitopes inA-protein were predetermined by performing the ELISA using variouscombinations of capture and conjugated enzymes with purified antigen(A-protein) to determine possible overlap of recognition sites.

The amount of color development was directly proportional to the amountof enzyme-linked antibody bound in the well, which is proportional tothe amount of antigen bound in the well by the “capture” antibody. Theresults were quantitated by a spectophotometric determination of theamount of color produced over a predetermined period of time (typically30 to 120 minutes). The ELISA described above has been used to testhuman serum from normal healthy volunteers, and cancer patients, for thepresence of the A-protein antigen. The normal population was used todetermine the threshold of positivity in this assay. The sensitivity ofthe assay extends at least to the single ng/ml range as determined bythe construction of standard sensitivity curves using known amounts ofpurified antigen.

The patient population tested in preliminary use of this assay yieldedthe following qualitative results. A positive reaction in the test wasobtained from patients diagnosed with lung, lymphoma, stomach, colon,rectal, and breast cancer when compared to normal subjects.

EXAMPLE 5

Preparation of Rabbit Polvclonal Antibodies that are Immunoreactive withthe Carboxyl Terminal Region of A-Protein

The following procedures were used to generate rabbit polyclonalantibodies that were immunoreactive with the carboxyl terminal portionof A-protein.

In one procedure, the carboxyl terminus 14 amino acid of the publishedsequence, QFEPQKVKEKMKNA (SEQ ID NO: 3), of human A-protein wassynthesized. A mixture of this synthetic peptide in Freunds adjuvant wasinjected subcutaneously at 3-4 separate sites in each rabbit. The amountof synthesized peptide in each injection was 50 μg. Two weeks later,boost injections of 50 μg synthesized peptide in Freunds adjuvant weregiven at 3-4 separate sites in each of the previously inoculatedanimals. After two weeks, test bleeds were made to assess blood antibodytiters with the synthesized peptide. Further boost injections were madeas needed to maintain adequate blood antibody titers. Sera from rabbitswith adequate blood antibody titers were collected and the desiredantibodies were obtained as a purified fraction by affinitychromatography techniques using the synthesized carboxyl terminuspeptide immobilized on a solid matrix. The purified antibodies wereprecipitated by dialysis in water and stored as a dry powder forsubsequent use. These rabbit polyclonal antibodies were reactive withthe peptide derived from the carboxyl terminal region of A-protein andwere designated as CY2A antibodies.

In another procedure, a peptide sequence of approximately 16 amino acidsat the carboxyl terminus of A-protein was synthetically produced. Thispeptide was then conjugated to the potent immunostimulator (adjuvant)molecule:keyhole limpet hemacyanin (KLH). The peptide-KLH conjugate wasthen injected into rabbits by standard procedures used to generateantibodies. The rabbits were subsequently bled and the sera were testedand shown to be positive for the A-protein carboxyl terminus peptideused in the immunogen. These rabbit polyclonal antibodies weredesignated as CPDD antibodies.

EXAMPLE 6

Assay for the Presence of A-Protein in a Liquid Sample

In order to detect the presence of A-protein in a specified sample, oneof the following procedures was performed:

A. Aliquots of a standard solution containing one of the monoclonalantibodies of Example 3 (the 3B9E1 antibodies) were placed in each wellof a 96 well titration tray and allowed to dry so the residualantibodies adhered to the bottom substratum of each well. Aliquots of a1% bovine serum albumin solution were added to each well for blockingpurposes. Residual liquid was removed. From each sample to be tested,50-100 μl of the sample were placed in specified wells and incubated forone hour at room temperature. The liquid portion was decanted and eachwell was washed with phosphate buffered saline (PBS). Aliquots of astandard solution containing the antibodies of Example 5 (the CY2Aantibodies), to which horseradish peroxidase had been linked, wereapplied to each well and incubated for one hour at room temperature. Theliquid portions were removed and each well was washed with PBS. Finally,aliquots of the horseradish peroxidase substrate,2,2′-azino-di-[3-ethyl-benzthiazoline sulfonate (6)] diammonium salt(ABTS), were added and development was allowed to occur for one hour at37° C. The degree of color development for each well was quantified byobtaining a spectrophotometric reading at 410 nm. These colormeasurements were converted to units by comparison to a calibrationcurve.

B. In another procedure, a biotin/streptavidin detection system wasutilized. The CPDD antibody of Example 5 was first conjugated to biotinusing covalent binding chemistry. An activated N-hydroxy-succinimideester of biotin was allowed to react with purified antibody so thatcovalent binding of the biotin molecules to primary amines on theantibody molecule occurred. Unreacted biotin was removed bychromatography. The antibody was then titered with the immunizingantigen to find the optimal assay dilution.

In general, the procedure was performed as follows:

The biotinylated antibody was first allowed to bind to its targetantigen. Simultaneously, a second antibody immobilized on a solid phasesupport (microtiter plate) captured the same antigen. After removal ofany unbound antibody by washing, the antibody:antigen:biotinylatedantibody complex was reacted with streptavidin conjugated to a reportermolecule, usually an enzyme such as horse radish peroxidase. Anotherwash step was performed to remove unbound streptavidin:enzyme. Asubstrate specific for the enzyme labeled streptavidin was then allowedto react with the remaining streptavidin:enzyme complex. The amount ofsubstrate hydrolyzed into the chromogenic product was thus directlyproportional to the amount of antigen present in the sample.

The advantage of the biotin/streptavidin detection system is that theantibody can be gently labeled with multiple biotin molecules withoutloss of antibody activity. The fact that more than one biotin is presenton each antibody molecule allows the signal to be amplified through thesubsequent binding of multiple streptavidin molecules.

Specifically, the procedure using the biotinylated antibodies wasperformed in the following manner:

Aliquots of a standard solution containing one of the monoclonalantibodies of Example 3 (the 3B9E1 antibodies) were placed in theindividual wells of a microtiter plate. The antibodies were allowed toadsorb to the bottom and sides of each well. The residual solutions wereaspirated from the wells and aliquots of a 1% bovine serum albuminsolution containing 20% sucrose were added to each well for blockingpurposes. Residual liquid was removed.

The subsequent reagents were initially allowed to warm to roomtemperature. Patient samples (EDTA plasma) were mixed and, if there wasany particulate matter observed, the sample was clarified bycentrifugation. A 50 μL aliquot of patient plasma and 200 μL of thebiotinylated CPDD rabbit polyclonal antibody in 0.05M phosphate bufferedsaline at pH 7.4 with 1 mg/mL bovine serum albumin were mixed and addedto the individual wells. These solutions were allowed to incubate in thewells for 2 hours. They were then aspirated and the wells were washed 3times with aspiration.

At this point in the procedure, 200 μL of streptavidin:horse radishperoxidase enzyme conjugate were added to each well and allowed toincubate for 1 hour at ambient temperature. The wells were then washedand aspirated 3 times as above. After this step, 200 μL oftetramethyl-benzidine substrate were pipetted into each well andincubated 20 minutes at ambient temperature. Then, 100 μL of stopsolution (0.05N sulfuric acid) were added. Absorbance for each well wasread on a dual wavelength microtiter plate spectrophotometer at 450/630nm. The mean absorbance for each sample was computed. (If duplicatesdisagreed by more than 10%, the sample was repeated.) Samples withabsorbance readings off scale were diluted and reassayed. The mean ofeach sample was divided by the mean of the negative controls to give aP/N value. Samples with a P/N greater than 2.0 were reported aspositive.

To establish the baseline for the negative control value (N), 40negative samples were initially assayed in duplicate to identify thosemost appropriate for establishing a cutoff between positive and negativesamples. A geometric mean was then calculated by eliminating the twohighest and lowest absorbance samples and recalculating the mean andstandard deviation of the remaining samples. The cutoff for positivitywas then arbitrarily set at 2 standard deviations above the mean for theremaining samples. Coincidentally, the mean plus 2 standard deviationswas very close to 2 times the mean. Hence, some assays used a cutoff ofpositivity of 2.0 where the unknown sample was divided by the mean ofthe negative control samples to establish a so-called P/N ratio.

Subsequently, a pool of negative human serum was obtained to serve asthe assay control/standard. This material had a mean absorbanceessentially identical to the geometric mean of the negative samplesabove. Thus, this negative control was calibrated against negative serato serve as the assay control. This assay control pool was run induplicate for each assay and the mean absorbance calculated with thecutoff for positivity stated as 2 times the mean of the negativecontrol.

EXAMPLE 7

Screening Procedure for Predicting Presence of Metastatic Cancer.

Blood samples from several individuals were collected. Included withinthe group of individuals were persons diagnosed as having metastaticbreast cancer, persons from a control population with no signs ofcancer, and persons that had previously been diagnosed as having hadcancer but, at the time of blood sampling, were diagnosed as havingtheir breast cancer in a state of remission.

A portion of the blood sample from each individual was used as thesample in the assay of Example 6A. The results of the assay for theblood sample from each individual is shown in Table 4.

TABLE 4 Assay Result Sample Diagnosis (ng A-protein/ml blood) gjscontrol 2.4 afb control 7.6 sac control 2.4 CY 02 NED(prostate) 1.8 CY08 control 1.6 2 control 3.5 3 control 9.7 6 control 8.8 7 control 2.8 8control 7.1 4 NED 4.7 1 NED 3.5 9 NED 3.8 5 NED 3.3 18  Mets; no chemo15.7 49  Mets; no chemo 3.7 132  Mets; no chemo 12.7 423  Mets; no chemo24.3 424  Mets; bgnchemo 26.4 515  Mets; no chemo 59.0 541  Mets; nochemo 22.3

The diagnosis labeled as NED refers to no evidence of diseaserecurrence. The results of this assay are also displayed in FIG. 1. Theaverage assay result for the combination of individuals identified ascontrols and persons whose cancer was in a state of remission is 4.5 ng.A-protein/ml blood. The average assay result for the individuals withmetastatic breast cancer is 23.4 ng A-protein/ml blood. With theexception of one individual who was diagnosed as having metastaticcancer but had an assay result within the control and NED range, allassay results could be clearly divided into two distinct groups: thoseindividuals who had no sign of cancer and those individuals who hadmetastatic breast cancer. Such a demarcation is unusual. The statisticalsignificance of these results was p<0.001 by a two-tailed t-test.

EXAMPLE 8

Screening Procedure for Predictins the Presence of Primary as Well asMetastatic Cancer

In an extensive screening procedure, blood plasma samples from overeight hundred human patients were subjected to the assay protocol ofExample 6B. The patient population included individuals with no cancer(controls), individuals with various stages of primary cancers,individuals with metastatic cancer and individuals with benign tumors.The screening procedure was conducted in a blinded fashion and theA-protein assay results were subsequently aligned with independentclinical diagnoses for purposes of assay verification.

The results of this screening procedure are shown in Table 5. The cancertype, cancer stage and number of individuals correctly assessed by theassay compared to the results of an independent diagnosis (sensitivity)are displayed.

TABLE 5 Cancer Type Assay Positives/Total # of Samples Breast 24/35 =69% Stage 1 1/6 2 3/4 4 1/2 ? 19/23 Primary Liver 17/25 = 68% Stage 21/1 3 5/7 3b 1/1 4 2/4 4a 7/9 4b 1/3 Pancreas 6/9 = 67% Stage 4 6/8 4a0/1 Prostate 24/41 = 61% Stage D2 0/2 ? 25/39 Bladder 11/19 = 58%Lymphoma 18/32 = 56% Stage ? 2/3 1 0/1 1a 1/1 1b 0/1 2a 1/4 2B 0/1 2bc1/1 2c 1/1 2ca 1/1 3b 3/4 4 3/6 4a 0/1 4B 4/4 4b 1/3 Head & Neck 9/16 =56% Stage 3 1/2 4  4/10 ? 4/4 Lung 41/91 = 45% Stage 1 4/5 2 2/4 3a 2/53b  6/13 4 10/27 ED 4/6 LD 2/7 ? 11/24 Colon & Rectal 17/43 = 40% Stage2 0/1 4 1/3 B1 2/2 B2 1/4 C2 2/4 D 3/3 ?  8/26 Leukemia 10/26 = 38%Multiple Myeloma 5/5 = 100% Stage 3a 3/3 3b 2/2 Endometrium 5/5 = 100%Stage 4 1/1 ? 4/4 Parotid 4/4 = 100% Cholangio CA 3/10 = 30% Stage 3 1/14 2/8 4a 0/1 Kidney 2/6 = 33% Stage 3 1/2 4 1/2 ? 0/2 Cervix 3/4 = 75%Stage 4 1/1 4a 1/1 ? 1/2 Thyroid 2/3 = 67% Stage 1 1/1 4 1/2 Brain 4/6 =67% Mouth 2/3 = 67% Uterus 2/7 = 29% Metastasis 2/7 = 29% (unknownorigin) Melanoma 2/2 = 100% Stage 4 2/2 Ovary 0/2 = 0% Abdominal 1/2 =50% Urinary 0/2 = 0% Tongue 1/3 = 33% Lip 1/1 = 100% Anal 1/1 = 100%Pelvic 1/1 = 100% Inguinal 1/1 = 100% Penile 1/1 = 100% Chest wall 1/1 =100% Fallopian Tube 1/1 = 100% Sarcomas 2/7 = 29% POEMS 1/1 = 100%Larynx 0/2 = 0% Germ Cell 0/1 = 0% Spinal Cord 0/1 = 0% Testicular 0/1 =0% Vulva 0/1 = 0% Spleen 0/1 = 0% Summary of All Cancers 262/518 = 51%

In addition, the assay was performed for normal (non-cancerous state)individuals and for persons with benign tumors. The results are shown inTable 6, where a “correct” assay result occurred (proper determinationthat the individual does not have cancer) when the P/N ratio for thatindividual was less than or equal to 2.0.

TABLE 6 Assay Result of Low Antigen Amount/Total No. of Samples Normals174/205 = 85% Benign Tumors  70/108 = 65%

The results of these assays demonstrate that the assay procedure hasmarked predictive ability to discern individuals with cancer whileeliminating from consideration those individuals who do not have cancer.The cancers detected by this assay include primary as well as metastaticcancers. Cancer in various stages, including stage 1 and stage 2, aredetected. In particular, breast cancer, prostate cancer, primary livercancer, lymphoma, pancreatic cancer, lung cancer, colon cancer, bladdercancer, endometrial cancer, and multiple myeloma are readily detectablewith the present assay.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

3 1 17 PRT bovine 1 Gly Asn Ser Lys Ser Gly Ala Leu Ser Lys Glu Ile LeuGlu Glu Leu 1 5 10 15 Gln 2 18 PRT bovine 2 Met Gly Asn Ser Lys Ser GlyAla Leu Ser Lys Glu Ile Leu Glu Glu 1 5 10 15 Leu Gln 3 14 PRT human 3Gln Phe Glu Pro Gln Lys Val Lys Gly Lys Met Lys Asn Ala 1 5 10

What is claimed is:
 1. A method of detecting the presence of cancer inan individual comprising: (a) obtaining a biological sample from saidindividual; (b) incubating said biological sample with at least oneantibody which is immunoreactive with A-protein; (c) detectingimmunoconjugates which form as a consequence of the incubation of step(b); and (d) relating the amount of immunoconjugates of step (c) to thepresence of cancer, wherein cancer is present when said amount isgreater than a threshold value.
 2. A method of detecting the presence ofa cancerous state in an individual comprising: (a) obtaining a liquidsample from said individual; (b) subjecting said liquid sample to anantibody sandwich assay wherein one of the two antibodies isimmunoreactive with a peptide that has a specified sequence ofapproximately 16 amino acids from the A-protein amino acid sequence, andthe other antibody is immunoreactive with a portion of A-protein otherthan said peptide with the specified approximately 16 amino acidA-protein sequence; (c) detecting the amount of antibody-A-proteinsandwich formed in step (b); and (d) relating said amount to thepresence of a cancerous state in said individual, wherein said cancerousstate is present when said amount is greater than a threshold value. 3.The method of claim 2 wherein at least one of said two antibodies is amonoclonal antibody.
 4. The method of claim 3 wherein the antibody thatis immunoreactive with a portion of A-protein other than said peptidewith the specified approximately 16 amino acid A-protein sequence is amonoclonal antibody.
 5. The method of claim 4 wherein the antibody thatis immunoreactive with a peptide that has a specified sequence ofapproximately 16 amino acids from the A-protein amino acid sequence is apolyclonal antibody with an attached detection label.
 6. The method ofclaim 2 wherein the liquid of said liquid sample is blood.
 7. The methodof claim 6 wherein: (i) the antibody that is immunoreactive with aportion of A-protein other than said peptide with the specifiedapproximately 16 amino acid A-protein sequence is a monoclonal antibodyadherent to a solid surface; and (ii) the antibody that isimmunoreactive with a peptide that has a specified sequence ofapproximately 16 amino acids from the A-protein amino acid sequence is apolyclonal antibody with an attached detection label.
 8. The method ofclaim 7 wherein said attached detection label is biotin.
 9. The methodof claim 7 wherein said threshold value is 2.0 times the mean of thenegative control value.
 10. The method of claim 9 wherein said cancerousstate is selected from the group consisting of breast cancer, prostatecancer, primary liver cancer, lymphoma, pancreatic cancer, lung cancer,colon cancer, bladder cancer, endometrial cancer and multiple myeloma.11. The method of claim 7 wherein said specified sequence ofapproximately 16 amino acids from the A-protein amino acid sequence iseither amino acids 142-158 of A-protein or the 16 amino acid carboxylterminus of A-protein.
 12. A method of detecting a cancer-diagnosticprotein, whose elevated level in the bloodstream of an individual ispredictive that the individual has cancer, comprising: (a) incubating aliquid sample from said individual with an antibody that immunoreactswith a peptide that has a specified sequence of approximately 16 aminoacids from the amino acid sequence of said cancer-diagnostic protein andwith an antibody that immunoreacts with a portion of saidcancer-diagnostic protein other than said peptide with the specifiedapproximately 16 amino acid cancer-diagnostic protein sequence, wherebyimmunocomplexes between said cancer-diagnostic protein and the twoantibodies are formed; and (b) detecting the amount of saidimmunocomplexes formed in step (a).
 13. The method of claim 12 whereinthe liquid of said liquid sample is blood.
 14. The method of claim 13wherein said liquid sample is incubated simultaneously with both saidantibodies.
 15. The method of claim 14 wherein: (i) said antibody thatimmunoreacts with a peptide that has a specified sequence ofapproximately 16 amino acids from the amino acid sequence of saidcancer-diagnostic protein is a polyclonal antibody with an attacheddetection label; and (ii) said antibody that immunoreacts with a portionof said cancer-diagnostic protein other than said peptide with thespecified approximately 16 amino acid cancer-diagnostic protein sequenceis a monoclonal antibody adherent to a solid surface.
 16. The method ofclaim 15 wherein said attached detection label is biotin.
 17. The methodof claim 13 wherein said liquid sample is incubated simultaneously withthe two said antibodies.
 18. The method of claim 13 wherein said liquidsample is first incubated with said antibody that immunoreacts with aportion of said cancer-diagnostic protein other than said peptide withthe specified approximately 16 amino acid cancer-diagnostic proteinsequence and then incubated with said antibody that immunoreacts with apeptide that has a specified sequence of approximately 16 amino acidsfrom the amino acid sequence of said cancer-diagnostic protein.
 19. Themethod of claim 18 wherein: (i) said antibody that immunoreacts with aportion of said cancer-diagnostic protein other than said peptide withthe specified approximately 16 amino acid cancer-diagnostic proteinsequence is a monoclonal antibody adherent to a solid surface; and (ii)said antibody that immunoreacts with a peptide that has a specifiedsequence of approximately 16 amino acids from the amino acid sequence ofsaid cancer-diagnostic protein is a polyclonal antibody with an attacheddetection label.
 20. The method of claim 15 wherein saidcancer-diagnostic protein is A-protein.
 21. The method of claim 20wherein an amount of said immunocomplexes greater than 2.0 times themean of the negative control value is predictive that said individualhas cancer.
 22. The method of claim 20 wherein said specified sequenceof approximately 16 amino acids from the amino acid sequence of saidcancer-diagnostic protein is either amino acids 142-158 of saidcancer-diagnostic protein or the 16 amino acid carboxyl terminus of saidcancer-diagnostic protein.
 23. A method of detecting the presence ofmetastatic cancer in an individual comprising: (a) obtaining abiological sample from said individual; (b) incubating said biologicalsample with at least one antibody which is immunoreactive withA-protein; (c) detecting immunoconjugates which form as a consequence ofthe incubation of step (b); wherein an amount greater than a thresholdvalue of said immunoconjugates detected in step c) is indicative of thepresence of metastatic cancer in said individual.
 24. The method ofclaim 23 wherein said biological sample is blood.
 25. The method ofclaim 24 wherein cancer is breast cancer.
 26. An antibody which binds toan approximately 16 amino acid sequence of A-protein to formimmunoconjugates whose presence in an amount greater than a thresholdvalue in a biological sample is predictive of the presence of cancer inthe individual from whom said biological sample was obtained, whereinsaid sequence of A-protein is selected from the group consisting of the14 amino acid carboxyl terminus, the 16 amino acid carboxyl terminus,the amino acid sequence 60-71, the amino acid sequence 142-158, and theamino acid sequence 158-170.
 27. The antibody of claim 26 wherein saidbiological sample is blood.
 28. A test kit for detecting the presence ofcancer in an individual comprising: (a) a first antibody thatimmunoreacts with a peptide that has a specified sequence ofapproximately 16 amino acids from the amino acid sequence of acancer-diagnostic protein; and (b) a second antibody that immunoreactswith a portion of said cancer-diagnostic protein other than said peptidewith the specified approximately 16 amino acid cancer-diagnostic proteinsequence.
 29. The test kit of claim 28 wherein either said firstantibody or said second antibody has an attached identifying label. 30.The test kit of claim 28 wherein at least one of said first antibody andsaid second antibody is a monoclonal antibody.
 31. The test kit of claim30 wherein said first antibody is a polyclonal antibody with an attachedidentifying label and said second antibody is a monoclonal antibodyadherent to a solid surface.
 32. The test kit of claim 31 wherein saidattached identifying label is biotin.
 33. The test kit of claim 28wherein said cancer-diagnostic protein is A-protein.
 34. The test kit ofclaim 33 wherein said specified sequence of approximately 16 amino acidsfrom the amino acid sequence of said cancer-diagnostic protein is eitheramino acids 142-158 of said cancer-diagnostic protein or the 16 aminoacid carboxyl terminus of said cancer-diagnostic protein.